Proteins having anticoagulant properties

ABSTRACT

Proteins which specifically inhibit coagulation Factor Xa. The inhibitors, which do not inhibit Factor VIIa, kallikrein, trypsin, chymotrypsin, thrombin, urokinase, tissue plasminogen activator, plasmin, elastase, Factor XIa or S. aureus V8 protease, are polypeptides of 60 amino acid residues. The inhibitors may be purified from Ornithodoros moubata extract, synthesized, or produced using a recombinant DNA yeast expression system.

This is a continuation of application Ser. No. 07/565,164, filed Aug.13, 1990 now abandoned, continuation in part of 07/498,307 filed Mar.23, 1990 now abandoned, continuation in part of 07/404,033 filed Sep. 7,1989, now abandoned.

BACKGROUND OF THE INVENTION

The role of blood coagulation is to provide an insoluble fibrin matrixfor consolidation and stabilization of a haemostatic plug. Formation ofa cross-linked fibrin clot results from a series of biochemicalinteractions involving a range of well-characterized plasma proteins.

The interactions are divided into what are termed the "intrinsicpathway", in which all the substances necessary for fibrin formation arepresent in precursor form in circulating plasma, and the extrinsicpathway in which thromboplastin, derived from tissues, bypasses severalsteps in the process and accelerates clot formation. The two pathwaysare highly interdependent and Factor VII, Factor IX and Factor X aremutually activated (J. C. Giddings "Molecular Genetics andImmunoanalysis in Blood Coagulation" Ellis Horwood Ltd., Chichester,England 1988, p. 17).

The role of Factor X in the coagulation cascade has been reviewed by Zuret al. "Tissue factor pathways of blood coagulation", Haemostasis andThrombosis, 1^(st) Edition (Bloom et al., Eds) Churchill Livingstone,Edinburgh, pp. 124-139 (1981); Jackson, "The biochemistry of prothrombinactivation" Haemostasis and Thrombosis, 2^(nd) edition (Bloom et al;Eds) Churchill Livingstone, Edinburgh, pp. 165-191 (1987) and Steinberget al. "The activation of Factor X" Haemostasis and Thrombosis, FirstEdition (Colman et al., Eds.) Lippincott, Philadelphia, pp. 91-111(1982).

Human Factor X circulates in plasma as a two-chain glycoprotein with amolecular weight of about 67000 estimated by gel electrophoresis in thepresence of sodium dodecylsulphate (Di Scipio et al., "A comparison ofhuman prothrombin, Factor IX (Christmas factor), Factor X (Stuartfactor) and protein S", Biochemistry, vol. 16, pp. 698-706). Slightlylower Mr values of about 59000 are obtained by sedimentation equilibriumanalysis. The normal plasma concentration is about 7-10 mg per liter andthe protein contains about 15% carbohydrate.

The heavy chain of Factor X, Mr=42000 demonstrates a high degree ofhomology with prethrombin 2 and contains the active site serine. It iscovalently linked to the light chain, Mr=17000, by disulphide bridges.The light chain contains the alpha-carboxylated glutamic acid residuesand shows significant homology with prothrombin fragment 1. Activationof Factor X by complexes of Factor IXa and Factor VIIIa or by tissuefactor/Factor VIIa involves at least two peptide cleavages. Theprincipal mechanism releases a small activation peptide from the heavychain of the molecule by hydrolysis of an arginine-isoleucine bond. Theactive product formed in this way is termed alpha-factor Xa. This ismodified further by cleavage of an arginine-glycine bond near thecarboxy terminus to form β-factor Xa. Both alpha-Xa and β-Xa have thesame coagulation activity.

The light chain of Factor X is unaffected by the activation process andremains linked to the heavy chain of the molecule by disulphide bridges.In this way the active substance retains the gamma-carboxyglutamic aciddomains necessary for calcium-mediate attachment to phospholipidmicelles or cellular surfaces. Fundamentally, therefore, in contrast tothrombin, Factor Xa remains associated with phospholipid and plateletmembranes.

U.S. Pat. No. 4,588,587 describes the anticoagulant activity ofHaementeria officinalis leech saliva.

Vermulen et al., Int. J. Biochem, Vol. 20, No. 6, pp. 621-31 (1988),describes the relative protease inhibition activities of tick toxinsisolated from R. evertsi evertsi, B. decoloratus, B. microplus, and H.truncatum. They were found to be fast-binding or slow-binding inhibitorsof trypsin and chymotrypsin.

Willadsen and Riding, Biochem J., Vol. 189, pp. 295-303 (1980), describeactivity of a proteolytic-enzyme inhibitor from the ectoparasitic tickBoophilus microplus, and the effect of the inhibitor onblood-coagulation parameters.

Prior studies identified and partially purified inhibitors of Factor XaMarkwardt, F. et al. (1958) Naturwissenschaften 45, pp, 398-399 andMarkwardt, F. et at. (1961) Naturwissenschaften 48 p. 433 and thrombinHawkins, R. et al. (1967) Proceedings of the Royal Society 70 p. in ticksaliva. It has been suggested that saliva contains an inhibitor ofFactor IXa (Hawkins). (Hellmann, K. and Hawkins (1967) Thromb. Diath.Haemorrh. 18 pp. 617-625) Ribeiro et al. reported that tick salivablocks clotting by inhibiting the intrinsic pathway (Ribeiro, J. (1985)J. Exp. Med. 161 pp. 332-344) but they did not identify the site ofinhibition. This study also demonstrated for the first time antiplateletactivity which blocked platelet aggregation induced by ADP, collagen, orplatelet activating factor (Ribeiro, J. (1985) J. Exp. Med. 161 pp.332-344). No attempt was made to purify any of these factors to enablean analysis of their structures or mechanism of action.

The S. cerevisiae α-mating factor pre-pro leader sequence has beenutilized in the expression of heterologous genes as secreted products inyeast (Brake et al., Proc. Natl. Acad. Sci. USA vol. 81, pp. 4642-4646(1984); Miyajima et al., Gene vol. 37, pp. 155-161 (1985); Vlasuk etal., J. Biol. Chem. vol. 261, pp. 4789-4796 (1986); Schultz et al., Genevol. 54, pp. 113-123 (1987); Schultz et al., Gene vol. 61, pp. 123-133(1987); Bayne et al., Gene, vol. 66 pp. 235-244 (1988); and Laison etal., Biotechnology, vol. 6, pp. 72-77 (1988)). Proteins produced asfusion products are proteolytically processed by the Lys-Arg-cleavingendopeptidase (KEX2) encoded by the KEX2 gene and products are secretedinto the culture medium. The KEX2 cleaves on the C-terminal side ofLys-Arg residues which are present between the ppL sequence and theheterologous gene.

SUMMARY OF THE INVENTION

The present invention includes proteins and related variants whichspecifically inhibit coagulation Factor Xa. The proteins are lowmolecular weight serine protease inhibitors. One of the proteins is asingle polypeptide of 60 amino acid residues with a calculated molecularmass of 6984.9. It is highly specific for Factor Xa (K_(i) =0.58 nM) anddoes not inhibit Factor VIIa, kallikrein, trypsin, chymotrypsin,thrombin, urokinase, plasmin, tissue plasminogen activator, elastase,Factor XIa or S. aureus V8 protease. The inhibitor does not requirecalcium. Complete amino acid sequences of these proteins were determinedand compared with other inhibitors of serine proteases. They havelimited homology with the Kunitz-type inhibitors. However, unlike otherknown inhibitors of this class, which all inhibit trypsin, it inhibitsFactor Xa almost exclusively.

The present invention also includes compositions comprising a protein ofthe invention which inhibit Factor Xa and prevent coagulation inpatients.

The present invention includes a method for obtaining a protein of theinvention from Ornithodoros moubata tick extracts.

The present invention also includes a method for transforming yeastcells with cloned DNA fragments introduced into yeast-E. coli shuttlevectors and grown in E. coli, and expressing a secreted protein of theinvention.

The present invention also includes a gene or degenerate equivalentencoding a protein of the present invention, and a method for producingthe protein in E. coli.

The present invention also includes methods for preparing a protein ofthe invention by synthetic means, and for expression vectors containingan appropriate genetic sequence for preparing a protein of the inventionusing recombinant technology and methods for preparing recombinantinhibitor from yeast cells using the expression vectors.

The present invention also includes methods for generating proteinmutants via PCR mutagenesis or Kunkel mutagenesis, thereby obtainingprotein variants having Factor Xa inhibiting activity.

The present invention also includes methods for treating a patient toinhibit blood coagulation comprising administering to the patient atherapeutically effective amount of a composition of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the reverse phase HPLC profiles of a human Factor Xainhibitor of the invention (TAP-1) following ion-exchange chromatography(A) of tick extracts and rechromatography of the active peak (bracketed)(B)

FIG. 2 shows SDS-PAGE of TAP-1 (Lane 1: inhibitor; Lane 2: molecularweight standards).

FIG. 3 shows the effect of increasing amounts of TAP-1 on the rate ofinitial velocities in the presence (Vi) and absence (Vo) of TAP-1following preincubation of TAP-1 with human Factor Xa in the absence ofsubstrate.

FIG. 4 shows insertion of α-mating factor•TAP gene to form pKH4•TAP, andspecific identification of KEX2 processing site.

DETAILED DESCRIPTION OF THE INVENTION

Proteins of the invention may be obtained from Ornithodoros moubata tickextracts, synthesized, or produced using recombinant technology.Preferred proteins of the invention have the following amino acidsequence: ##STR1## wherein AA₁, AA₂, AA₃ and AA₄, are Arg, Tyr, Gly andArg respectively, (referred to hereinafter as "TAP-1") or AA₁, AA₂, AA₃and AA₄ are Gln, Phe, Asp and Gln respectively (referred to hereinafteras "TAP-2")

Also preferred are variants of TAP-1 having one or more of the arginineamino acids at positions 9, 23, 27 and 53 replaced by asparagine, andvariants of TAP-2 having one or more of the arginine amino acids atpositions 23 and 27 replaced by asparagine.

Also preferred are variants of TAP-1 having aspartic acid at position 16replaced by arginine.

Also within the present invention are bifunctional proteins having apolypeptide sequence comprising the above-described sequence havingTAP-1 protein activity and included therein the tripeptide sequencearginine-glycine-aspartic acid (RGD). The presence of the RGD tripeptideis achieved, for example, by inserting glycine between amino acidpositions 9 and 10, wherein AA₁ is arginine, or between amino acidpositions 53 and 54, wherein AA₄ is arginine.

The bifunctional molecules are particularly suited for targetingproteins of the present invention to activated platelets where there isan assembled prothrombinase complex. The prothrombinase complex,composed of Factor Xa, Factor Va, an appropriate phospholipid surfaceand prothrombin, is responsible for the Factor Xa conversion ofprothrombin to thrombin in vivo. The phospholipid component of thiscomplex can be supplied by any cellular surface. However, it has beendemonstrated that upon activation, platelets vesicularize intomicroparticles which expose Factor Va binding sites. These activatedplatelet micro-particles are extremely active in supporting theprothrombinase complex. Insertion of an RGD sequence in an appropriatelocation of a non-adhesive protein creates a protein capable of bindingto integrins which recognize this peptide sequence such as theglycoprotein IIb/IIIa protein (Maeda, T. et al. (1989), J. Biol. Chem.264, 15165-15168). Upon activation, the platelet IIb/IIIa complexbecomes exposed and is critical in the subsequent fibrinogen-mediatedaggregation. Insertion of an RGD sequence in an appropriate region ofTAP (by, for example, site-specific mutagenesis) creates a bifunctionalmolecule targeted to activated platelets where the prothrombinasecomplex is assembled.

The K_(i) for inhibition of TAP-1 is 0.58 nM.

Proteins of the present invention and their isoforms and naturalvariants are obtained by:

(a) homogenizing Ornithodoros moubata tick extracts, centrifuging thehomogenate to produce supernatant protein suspension fractions andlyophilizing the fractions;

(b) dissolving the lyophilized fractions in water and applying thesolution to a gel filtration chromatography column to isolate thefractions containing Factor Xa inhibitory activity; and

(c) applying the fractions containing Factor Xa inhibitory activity toan anion exchange column eluted with a NaCl gradient to collectfractions which inhibit Factor Xa.

The invention encompasses all natural homologues, isoforms or geneticvariants having highly specific inhibitory activity against Factor Xasuch as the activity of TAP-1 and TAP-2.

Proteins

Proteins of the invention include variations on the disclosed purifiedprotein sequence or sequences which conserve the activity of thedisclosed sequence or sequences, including fragments or subunits,naturally occurring mutations, allelic variants, randomly generatedartificial mutants and intentional sequence variation which conservesactivity. Fragments or subunits refers to any portion of the sequencewhich contains fewer amino acids than the complete protein, e.g. partialsequences excluding portions at the N- and/or C-termini of the completeprotein.

Proteins of the invention also include disclosed recombinant proteinsequence or sequences which conserve the activity of the purifiedprotein sequence or sequences. Also included are hybrid proteins, suchas fusion proteins or proteins resulting from the expression of multiplegenes within the expression vector, and may include a polypeptide havingthe specific activity of a disclosed protein linked by peptide bonds toa second polypeptide.

It will be understood that other variants of any of the native proteinsof the present invention are included, especially any variants thatdiffer from the isolated proteins only by conservative amino acidsubstitution. Conservative amino acid substitutions are defined as"sets" in Table 1 of Taylor, W. R., J. Mol. Biol., Vol. 188, p. 233(1986). The proteins or fragments thereof in this application includeany such variations in the amino acid substitution, deletion, or otherprocess, provided that the protein, after purification, immunochemicallyreacts with antibodies specific for the above-described inhibitorproteins.

Proteins of the invention may be prepared using solid phase synthesis,such as that described by Merrifield, J. Am. Chem. Soc., 85, 2149 (1964)or other equivalent chemical syntheses known in the art such as thesyntheses of Houghten, Proc. Natl. Acal. Sci., 82, 5132 (1985), payingparticular attention to treatment of the protein-containing solutionfollowing HF cleavage. Solid-phase synthesis is commenced from theC-terminus of the peptide by coupling a protected amino acid to asuitable resin, as generally set forth in U.S. Pat. No. 4,244,946,issued Jan. 21, 1982 to Rivier et al., the disclosure of which is herebyincorporated by reference. Examples of synthesis of this general typeare set forth in U.S. Pat. Nos. 4,305,872 and 4,316,891.

In synthesizing the polypeptides, the carboxyl terminal amino acid,having its alpha-amino group suitable protected, is coupled to achloromethylated polystyrene resin or the like. After removal of thealpha-amino protecting group, as by using trifluoroacetic acid inmethylene chloride, the next step in the synthesis is ready to proceed.Other standard cleaving reagents and conditions for the removal ofspecific amino protecting groups may be used, as described in the openliterature.

The remaining alpha-amino- and side-chain-protected amino acids are thencoupled stepwise in the desired order by condensation to obtain anintermediate compound connected to the resin. As an alternative toadding each amino acid separately in the synthesis, some of them may becoupled to one another prior to the addition to the growing solid-phasechain. The selection of the appropriate coupling reagents is within theskill of the art.

The condensation between two amino acids, or an amino acid and apeptide, or a peptide and a peptide can be carried out according to theusual condensation methods such as axide method, mixed acid anhydridemethod, DCC (dicyclohexylcarbodiimide) method, active ester method(p-nitrophenyl ester method, BOP [benzotriazole-1-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate] method,N-hydroxysuccinic acid imido ester method, etc), Woodward reagent Kmethod. In the case of elongating the peptide chain in the solid phasemethod, the peptide is attached to an insoluble carrier at theC-terminal amino acid. For insoluble carriers, those which react withthe carboxy group of the C-terminal amino acid to form a bond which isreadily cleaved later, for example, halomethyl resin such aschloromethyl resin and bromomethyl resin, hydroxymethyl resin,aminomethyl resin, benzhydrylamine resin, andt-alkyloxycarbonylhydrazide resin can be used.

Common to chemical syntheses of peptides is the protection of thereactive side-chain groups of the various amino acid moieties withsuitable protecting groups at that site until the group is ultimatelyremoved after the chain has been completely assembled. Also common isthe protection of the alpha-amino group on an amino acid or a fragmentwhile that entity reacts at the carboxyl group followed by the selectiveremoval of the alpha-amino-protecting group to allow subsequent reactionto take place at that location. Accordingly, it is common that, as astep in the synthesis, an intermediate compound is produced whichincludes each of the amino acid residues located in the desired sequencein the peptide chain with various of these residues having side-chainprotecting groups. These protecting groups are then commonly removedsubstantially at the same time so as to produce the desired resultantproduct following purification.

The applicable protective groups for protecting the alpha- andomega-side chain amino groups are exemplified such as benzyloxycarbonyl(hereinafter abbreviated as Z), isonicotinyloxycarbonyl (iNOC),O-chlorobenzyloxycarbonyl [Z(2Cl], p-nitrobenzyloxycarbonyl [Z(NO₂ ],p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl, (Boc),t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamatyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl(Fmoc), methylsulfonylethoxycarbonyl (Msc), trifluoroacetyl, phthalyl,formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt),dimethylphosphinothioyl (Mpt) and the like.

As protective groups for carboxy group there can be exemplified, forexample, benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzylester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and thelike. It is desirable that specific amino acids such as arginine,cysteine, and serine possessing a functional group other than amino andcarboxyl groups are protected by a suitable protective group as occasiondemands. For example, the guanidino group in arginine may be protectedwith nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl,p-methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Mds),1, 3, 5-trimethylphenysulfonyl (Mts), and the like. The thiol group incysteine may be protected with p-methoxybenzyl, triphenylmethyl,acetylaminomethyl, ethylcarbamoyle, 4-methylbenzyl, 2, 4,6-trimethybenzyl (Tmb) etc, and the hydroxyl group in serine can beprotected with benzyl, t-butyl, acetyl, tetrahydropyranyl etc.

Stewart and Young, "Solid Phase Peptide Synthesis", Pierce ChemicalCompany, Rockford, Ill. (1984) provides detailed information regardingprocedures for preparing peptides. Protection of α-amino groups isdescribed on pages 14-18, and side-chain blockage is described on pages18-28. A table of protecting groups for amine, hydroxyl and sulfhydrylfunctions is provided on pages 149-151. These descriptions are herebyincorporated by reference.

After the desired amino-acid sequence has been completed, theintermediate peptide is removed from the resin support by treatment witha reagent, such as liquid HF and one or more thio-containing scavengers,which not only cleaves the peptide from the resin, but also cleaves allthe remaining side-chain protecting groups. Following HF cleavage, theprotein sequence is washed with ether, transferred to a large volume ofdilute acetic acid, and stirred at pH adjusted to about 8.0 withammonium hydroxide.

Preferably in order to avoid alkylation of residues in the polypeptide,(for example, alkylation of methionine, cysteine, and tyrosine residues)a thio-cresol and cresol scavenger mixture is used. The resin is washedwith ether, and immediately transferred to a large volume of diluteacetic acid to solubilize and minimize intermolecular cross-linking. A250 μM polypeptide concentration is diluted in about 2 liters of 0.1Macetic acid solution. The solution is then stirred and its pH adjustedto about 8.0 using ammonium hydroxide. Upon pH adjustment, thepolypeptide takes its desired conformational arrangement.

Recombinant DNA Technology

Recombinant DNA technology may be used to produce proteins of theinvention. This technology allows segments of genetic information, DNA,from different cells, and usually from different organisms, to be joinedend-to-end outside the organisms from which the DNA was obtained and toincorporate this hybrid DNA into a cell that will allow the productionof the protein for which the original DNA encodes. Genetic information,DNA or mRNA, is isolated and incorporated into an appropriate cloningvector, and transduced into an appropriate host cell.

Cloning vectors useful for this technology include a DNA sequence whichaccommodates specific experimental foreign DNA. The vectors areintroduced into host cells that can exist in a stable manner and expressthe protein dictated by the experimental DNA. Cloning vectors mayinclude plasmids, bacteriophage, viruses and cosmids.

Expression vectors are DNA sequences that are required for thetranscription of cloned copies of genes and the translation of theirmRNAs in an appropriate host. These vectors can express eitherprocaryotic or eucaryotic genes in a variety of cells such as bacteria,yeast, insect and mammalian cells.

Proteins may also be expressed in a number of virus systems. A suitablyconstructed expression vector contains an origin of replication forautonomous replication in host cells, selective markers, a limitednumber of useful restriction enzyme sites, a high copy number, andstrong promoters. Promoters are DNA sequences that direct RNA polymeraseto bind to DNA and initiate RNA synthesis; strong promoters cause suchinitiation at high frequency. Expression vectors may include, but arenot limited to cloning vectors, modified cloning vectors andspecifically designed plasmids or viruses.

Expression Systems

Procaryotes most frequently are represented by various strains of E.coli. Other microbial strains may be used, such as bacilli, e.g.Bacillus subtilis, various species of Pseudomonas, or other bacterialstrains. In such procaryotic systems, plasmid vectors which containreplication sites and control sequences derived from a speciescompatible with the host are used. For example, E. coli is typicallytransformed using derivatives of pBR322, a plasmid derived from an E.coli species by Bolivar et al., Gene (1977) 2:95. Commonly usedprocaryotic control sequences, which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, include such commonly usedpromoters as the beta-lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al., Nature (1977) 198:1056) and thetryptophan (Trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8:4057) and the lambda-derived P_(L) promoter and N-gene ribosomebinding site (Shimatake et al., Nature (1981) 292:128). However, anyavailable promoter system compatible with procaryotes can be used.

Expression systems useful in the eucaryotic systems of the inventioncomprise promoters derived from appropriate eucaryotic genes. A class ofpromoters useful in yeast, for example, include promoters for synthesisof glycolytic enzymes, including those for 3-phosphoglycerate kinase(Hitzeman et al., J. Biol. Chem. (1980) 255:2073). Other promotersinclude those from the enolase gene (Holland, M. J., et al., J. Biol.Chem. (1981) 256:1385) or the Leu2 gene obtained from YEp13 (Broach, J.,et al., Gene (1978) 8:121).

Suitable mammalian promoters including the early and late promoters fromSV40 (Fiers, et al., Nature (1978) 273:113) or other viral promoterssuch as those derived from polyoma, adenovirus II, bovine papillomavirus or avian sarcoma viruses. Suitable viral and mammalian enhancersare cited above. In the event plant cells are used as an expressionsystem, the nopaline synthesis promoter is appropriate (Depicker, A. etal., J. Mol. Appl. Gen. (1982) 1:561).

Insect cell expression systems useful for expressing the proteinsinclude modified versions of the system described in Smith et al., U.S.Pat. No. 4,745,051. Baculovirus DNA comprising a baculovirus gene or aportion thereof which includes a promoter of the baculovirus gene iscleaved to obtain a DNA fragment containing at least the promoter. Thedesired product protein is prepared by infecting a susceptible hostinsect cell with a recombinant baculovirus expression vector wherein theexpression vector is a recombinant baculovirus genome comprising atleast one selected heterologous product protein polypeptide structuralgene under the transcriptional control of a baculovirus polyhedrinpromoter.

A recombinant baculovirus expression vector capable of expressing aselected gene in a host insect cell is produced by cleaving baculovirusDNA to produce a DNA fragment comprising a baculovirus polyhedrinpromoter, and sufficient flanking DNA sequences to facilitate homologousrecombination; inserting the baculovirus DNA fragment into a cloningvehicle to form a modified cloning vector; identifying a selectedrestriction site of the cloned baculovirus DNA fragment which is underthe transcriptional control of the baculovirus polyhedrin promoter;deleting from the modified cloning vector the additional restrictionsite in the baculovirus DNA fragment under the transcriptional controlof the baculovirus polyhedrin promoter; inserting a selectedheterologous gene into the unique restriction site to form a recombinantshuttle vector; contacting the baculovirus DNA so as to effectrecombination, thereby producing a mixture of recombinant andnonrecombinant baculoviruses; and isolating a recombinant baculovirusexpression vector from the mixture.

Oligonucleotide Primers

Oligonucleotide primers are prepared which will hybridize to differentstrands of the desired sequence and at relative positions along thesequence such that an extension product synthesized from one primer,when it is separated from its template (complement), can serve as atemplate for extension of the other primer into a nucleic acid ofdefined length. The primers may be prepared using any suitable method,such as, for example, the phosphotriester and phosphodiester methods,described respectively in Narang, S. A., et al. Meth. Enzymol., 68, 90(1979) and Brown, E. L. et al., Meth. Enzymol, 68, 109 (1979), orautomated embodiments thereof. In one such automated embodiment,diethylphosphoramidites are used as starting materials and may besynthesized as described by Beaucage et al., Tetrahedron Letters (1981),22: 1859-1862. One method for synthesizing oligonucleotides on amodified solid support is described in U.S. Pat. No. 4,458,066. It isalso possible to use a primer which has been isolated from a biologicalsource (such as a restriction endonuclease digest).

Probing cDNA Libraries

cDNA or genomic libraries are screened using the colony or plaquehybridization procedure. Each plate containing bacterial colonies (orrecombinant phage-infected bacteria) is replicated onto duplicatenitrocellulose filter papers (S & S type BA-85) and, for bacterialcolony screens, the colonies are allowed to grow at 37° C. for 14-16hours on L agar containing 50 μg/ml Amp. The bacteria are lysed plasmidor phage and DNA fixed to the filter by sequential treatment for 5minutes each time with 0.2N NaOH, 1.5M NaCl, then 0.5M Tris pH 7.5, 1.5MNaCl and then 2×standard saline citrate (2×SSC). Filters are air driedand baked at 80° C. for 2 hours. The duplicate filters are prehybridizedat 42° C. for 6-8 hours with 10 ml per filter of DNA hybridizationbuffer (5×SSC, pH 7.0, 5×Denhardt's solution (polyvinyl pyrrolidine,plus Ficoll and bovine serum albumin; 1×=0.02% of each), 50 mM sodiumphosphate buffer at pH 7.0, 0.2% SDS, 20 μg/ml polyU, and 50 μg/mldenatured salmon sperm DNA.

The samples are hybridized with kinased probe under conditions whichdepend on the stringency desired. Typical moderately stringentconditions employ a temperature of 42° C. for 24-36 hours with 1-5ml/filter of DNA hybridization buffer containing probe. For higherstringencies, high temperatures and shorter times are employed. Thefilters are washed four times for 30 minutes each time at 37° C. with2×SSC, 0.2% SDS and 50 mM sodium phosphate buffer at pH 7, then arewashed twice with 2×SSC and 0.2% SDS, air dried and are autoradiographedat -70° C. for 2 to 3 days.

Polymerase Chain Reaction Amplification

Large amounts of DNA coding for the protein may be obtained usingpolymerase chain reaction (PCR) amplification techniques as described inMullis et al., U.S. Pat. No. 4,800,159. The extension product of oneprimer, when hybridized to another primer, becomes a template for theproduction of the nucleic acid sequence.

The nucleic acid sequence strands are heated until they separate, in thepresence of oligonucleotide primers that bind to their complementarystrand at a particular site of the template. The primer templatecomplexes act as substrate for DNA polymerase which, in performing itsreplication function, extends the primers. The region in common withboth primer extensions, upon denaturation, serves as template for arepeated primer extension. This process is continued with a series ofheating and cooling cycles, heating to separate strands, and cooling toreanneal and extend the sequences. More and more copies of the strandsare generated as the cycle is repeated. Through amplification, thecoding domain and any additional primer-encoded information such asrestriction sites or translation signals (signal sequences, start codonsand/or stop codons) is obtained.

Vector Construction

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g. New England Biolab s, Product Catalog. In general, about 1 μg ofplasmid or DNA sequence is cleaved by one unit of enzyme in about 20 μlof buffer solution. Typically, an excess of restriction enzyme is usedto ensure complete digestion of the DNA substrate. Incubation times ofabout 1 to 2 hours at about 37° C. are workable, although variations canbe tolerated. After each incubation, protein is removed by extractionwith phenol/chloroform, and may be followed by running over a Sephadex®G-50 spin column. If desired, size separation of the cleaved fragmentsmay be performed by polyacrylamide gel or agarose gel electrophoresisusing standard techniques. A general description of size separations isformed in Methods in Enzymology (1980)65: 499-560.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 minutes at 20 to 25° C. in 50 mM Tris, pH 7.6, 50 mMNaCl, 6 mM MgCl₂, 6 mM DTT and 5-10 μMdNTPs. The Klenow fragment fillsin 5' overhangs but removes protruding 3' single strands, even in thepresence of the four dNTPs. If desired, selective repair can beperformed by supplying only one of the, or selected, dNTPs within thelimitations dictated by the nature of the sticky ends. After treatmentwith Klenow, the mixture is extracted with phenol/chloroform and ethanolprecipitated followed by running over a Sephadex® G-50 spin column.Treatment under appropriate conditions with S1 nuclease results inhydrolysis of any single-stranded portion.

As mentioned above, oligonucleotides may be prepared by the triestermethod of Matteucci, et al. (J. Am. Chem. Soc. (1981) 103:3185) or usingcommercially available automated oligonucleotide synthesizers. Kinasingof single strands prior to annealing or for labelling is achieved usingan excess, e.g., approximately 10 units of polynucleotide kinase to 0.1nmole substrate in the Presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mMdithiothreitol, 1-2 mM ATP, 1.7 pmoles ³² P-ATP(2.9 mCi/mmole), 0.1 mMspermidine, 0.1 mM EDTA

Ligations are performed in 15-30 μl volumes under the following standardconditions and temperatures: 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and 1 mM ATP, 0.3-0.6 (Weiss) unitsT4 DNA ligase at 14° C. (for "blunt end" ligation). Intermolecular"sticky end" ligations are usually performed at 33-100 μg/ml total DNAconcentrations (5-100 nM total end concentration). Intermolecular bluntend ligations (usually employing a 10-30 fold molar excess of linkers)are performed at 1 μM total ends concentration.

In vector construction employing "vector fragments," the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) in orderto remove the 5' phosphate and prevent religation of the vector. BAPdigestions are conducted at pH 8 in approximately 150 mM Tris, in thepresence of Na⁺ and Mg²⁺ using about 1 unit of BAP per μg of vector at60° C. for about 1 hour. In order to recover the nucleic acid fragments,the preparation is extracted with phenol/chloroform and ethanolprecipitated and desalted by application to a Sephadex® G-50 spincolumn. Alternatively, religation can be prevented in vectors which havebeen double digested by additional restriction enzyme digestion of theunwanted fragments.

For portions of vectors derived from cDNA or genomic DNA which requiresequence modifications, site specific primer directed mutagenesis isused. This is conducted using a primer synthetic oligonucleotidecomplementary to a single stranded plasmid or phage DNA to bemutagenized except for limited mismatching, representing the desiredmutation. Briefly, the synthetic oligonucleotide is used as a primer todirect synthesis of a strand complementary to the phage, and theresulting double-stranded DNA is transformed into a phage-supportinghost bacterium. Cultures of the transformed bacteria are plated in topagar, permitting plaque formation from single cells which harbor thephage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.The resulting plaques are hybridized with kinased synthetic primer at atemperature which permits hybridization of an exact match, but at whichthe mismatches with the original strand are sufficient to preventhybridization. Plaques which hybridize with the probe are then picked,cultured, and the DNA recovered.

Verification of Construction

In the constructions set forth below, correct ligations for plasmidconstruction are confirmed by first transforming E. coli strain MM294obtained from E. coli Genetic Stock Center, CGSC #6135, or othersuitable host with the ligation mixture. Successful transformants areselected by ampicillin, tetracycline or other antibiotic resistance orusing other markers depending on the mode of plasmid construction, as isunderstood in the art. Plasmids from the transformants are then preparedaccording to the method of Clewell, D. B., et al, Proc. Natl. Acad. Sci.USA (1969) 62:1159, optionally following chloramphenicol amplification(Clewell, D. B., J. Bacteriol (1972) 110:667). The isolated DNA isanalyzed by restriction and/or sequenced by the dideoxy method ofSanger, F., et al, Proc. Natl. Acad. Sci. USA (1977) 74:5463 as furtherdescribed by Messing, et al, Nucleic Acids Res (1981) 9:309, or by themethod of Maxam, et al, Methods in Enzymology (1980) 65:499.

Transformation

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S.N., Proc. Natl. Acad. Sci.USA (1972) 69:2110, or the RbCl method described in Maniatis et al.,Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Press,p. 254 is used for procaryotes or other cells which contain substantialcell wall barriers. Infection with Agrobacterium tumefaciens (Shaw, C.H., et al., Gene (1983) 23:315) is used for certain plant cells. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology (1978) 52:546 ispreferred. Transformations into yeast are carried out according to themethod of Van Solingen, P., et al., J. Bacter. (1977) 130:946 and Hsiao,C. L. et al., Proc. Natl. Acad. Sci. USA (1979) 76:3829.

A synthetic gene coding for the inhibitor was made usingoligonucleotides. The synthetic gene was fused to cheY to make a fusionprotein in E. coli, and also fused to ompA signal peptide for secretionof the protein into the E. coli periplasm.

EXAMPLE 1

Ornithodoros moubata ticks were obtained from South Africa throughAntibody Associates, Inc. (Bedford, Tex.). Colorimetric substrates werepurchased from Helena Labs, American Diagnostica, and Chemical Dynamics.Human Factor Xa was from Enzyme Research Laboratories. Human plasmin,urokinase, and bovine thrombin were from Calbiochem; bovine trypsin,chymotrypsin, and pancreatic elastase were from Worthington Enzymes,Inc. Plasma kallikrein, carboxypeptidase Y, S. aureus V8 protease andprotease inhibitors were from Sigma. Two chain tPA was from AmericanDiagnostica.

Preparation of Crude Extracts

Fifty ticks (0.8 g) were homogenized in a ground glass homogenizer in 3ml 20 mM Bis-Tris (pH 7.0) containing 0.15M NaCl and 50 uM E-64, 50 uMpepstatin, and 50 uM chymostatin. The homogenate was centrifuged 30 min.at 30,000×g and the resultant pellet was resuspended in 3 ml buffer andrecentrifuged. The supernatants were combined, sucrose was added to afinal concentration of 10 mg/ml and the extract was lyophilized. Thismaterial was dissolved in 2 ml of H₂ O and applied to a column ofSephadex G-75 superfine (Pharmacia) (1.5 cm×95 cm) equilibrated in 20 mMBis-Tris-HCl (pH 7.4) containing 25 mM NaCl and 0.1 mM EDTA. Two mlfractions were collected and aliquots from selected fractions wereassayed for their effect on Factor Xa and thrombin.

Purification of the Inhibitor of Factor Xa

After gel filtration, the fractions containing Factor Xa inhibitoryactivity were pooled and applied to a column of Mono Q (Pharmacia)(0.5×5 cm) equilibrated in 20 mM Bis-Tris-HCl (pH 6.0). The column waseluted with a gradient of NaCl (0 to 1M NaCl; 60 ml total), and 1 mlfractions were collected. The fractions which inhibited Factor Xa weredialyzed to remove salt and lyophilized. This material was dissolved in2 ml of H₂ O and was applied to a Vydac C₁₈ column. (4.6×200 mm)equilibrated in 0.1% trifluoroacetic acid. The proteins were eluted witha linear gradient of acetonitrile (0 to 60%) in 0.1% trifluoroaceticacid at 1% per min. One ml fractions were collected and the solventsremoved under vacuum. Fractions were dissolved in 0.5 ml 20 mM Tris-HCl(pH 7.4)/.15M NaCl (TBS) and assayed. Those containing inhibitoryactivity were pooled and rechromatographed on the same column with aconvex gradient of increasing acetonitrile (0 to 40%). The peaks werecollected by hand and dried down.

A crude soluble extract of whole ticks was fractionated by gelfiltration on Sephadex G-75. When selected column fractions were assayedfor their ability to inhibit Factor Xa, one peak was found which elutedwith an apparent molecular weight of 8,000-10,000.

The peak of Factor Xa inhibitory activity was pooled and applieddirectly to a column of Mono Q. The inhibitor eluted in one peak of0.25M NaCl. In an earlier attempt to purify this inhibitor a second peakof activity was found which eluted at a lower ionic strength. It waspurified by similar procedures (see below).

This material was dialyzed to remove salt and lyophilized. Afterredissolving in a small volume of H₂ O, aliquots were fractionated byreverse phase HPLC. The inhibitory activity eluted in one peak (FIG.1A). This material was rechromatographed to obtain a homogeneous peak ofprotein (FIG. 1B). We estimate that 200-250 ug of the purified inhibitorcould be obtained from 500 ticks. On SDS-PAGE one band of protein wasfound which had an estimated Mr=6000 (FIG. 2).

Other Techniques

Protein was estimated by the Lowry procedure using bovine serum albuminas a standard. SDS-PAGE was carried out on slab gels containing 16%acrylamide and 0.5% bisacrylamide. Pre-stained molecular weightstandards were obtained from BRL. Proteins were detected by stainingwith Coomassie blue.

Enzyme Assays

Assays were carried out at room temperature in 96-well microtiterplates. The color developed from the hydrolysis of peptide-nitroanilidesubstrates was monitored continuously at 405 nM on a V_(max) machine(Molecular Devices). The concentration of Factor Xa was determined byactive site titration. The concentrations of working solutions of theother proteases were determined spectrophotometrically using publishedextinction coefficients. The concentration of purified inhibitor wasdetermined by quantitative amino acid analysis. Typically, the assayincluded 500 pM proteolytic enzyme, 20 mM Tris-HCl (pH 7.4), 0.15M NaCl,0.2-0.3 mM substrate and an aliquot of selected column fractions or thepurified inhibitor in a total volume of 200-220 ul. In the case of humanFactor Xa, the buffers also included 0.1% BSA; for S. aureus V8 proteasethe buffer was 50 mM NH₄ HCo₃. The substrates used were: Spectrozyme Xa(Factor Xa), S-2238 (thrombin), S-2356 (chymotrypsin), S-2366 (FactorXIa) Boc-AlaAla-Pro-Ala-pNA (elastase), Spectrozyme Pkal (kallikrein),Spectrozyme PL (plasmin), Spectrozyme UK (urokinase) Z-Phe-Leu-Glu-pNA(S. aureus V8 protease) and Spectrozyme tPA for tPA. Factor VIIa wasassayed by measuring the release of the ³ H-activation peptide fromhuman Factor X. For kinetic analyses, assays were carried for up to 10min. followed by estimation of the initial rate where less than 5% ofthe added substrate was utilized.

Structure Determination

Prior to digestion with proteases or sequence analysis, the inhibitorwas denatured by incubating for 1 hour at 37° C. in 6M guanidinehydrochloride containing 50 mM dithiothreitol, 0.25M Tris-HCl (pH 8.2)and 1 mM EDTA. Iodoacetamide was added to a final concentration of 0.1Mand the mixture was allowed to stand at room temperature for 30 min. toblock the sulfhydryl groups. The protein was reisolated by applying thereaction mixture directly to a Vydac C₁₈ column and eluting it with agradient of acetonitrile in 0.1% trifluoroacetic acid. The protein wasdried down under vacuum and sequenced directly on an Applied Biosystemsgas phase sequenator.

The reduced and alkylated inhibitor was dissolved in 100 ul 50 mM NH₄HCO₃ and incubated at room temperature with trypsin (50:1, w/w). After 4hours, the same amount of trypsin was added and the reaction was allowedto proceed overnight. The mixture was then applied directly to a C₁₈column to fractionate the digestion products. Proteolysis by S. aureusV8 protease was carried out similarly except that the buffer was 50 mMammonium acetate. The peaks were collected by hand and submitted forsequence analysis.

After reduction and carboxyamidomethylation, the inhibitor was sequenceddirectly. As shown in Table I, the sequence of the first 53 residues wasdetermined in this way, although the identity of several amino acids wasambiguous. To extend this sequence, reduced and alkylated protein wastreated with trypsin and the fragments separated by reverse phase HPLC.The polypeptides sequence was found to be the sequence of TAP-1.

                  TABLE I                                                         ______________________________________                                        NH.sub.2 -Terminal Sequence of RCM Inhibitor                                  Cycle         PTH        Yield                                                No.           Amino Acid (pmol)                                               ______________________________________                                         1            Tyr        488                                                   2            Asn        565                                                   3            Arg        202                                                   4            Leu        891                                                   5            Cm--Cys    461                                                   6            Ile        655                                                   7            Lys        426                                                   8            Pro        415                                                   9            Arg        247                                                  10            Asp        326                                                  11            Trp        149                                                  12            Ile        368                                                  13            Asp                                                             14            Glu                                                             15            Cm--Cys                                                         16            Asp                                                             17            Ser                                                             18            Asn                                                             19            Glu                                                             20            Gly                                                             21            Gly                                                             22            Glu                                                             23            Arg                                                             24            Ala                                                             25            Tyr                                                             26            Phe                                                             27            Arg                                                             28            Asn                                                             29            Gly                                                             30            Lys                                                             31            Gly                                                             32            Gly                                                             33            Cm--Cys                                                         34            Asp                                                             35            Ser                                                             36            Phe                                                             37            Trp                                                             38            Ile                                                             39            Cm--Cys                                                         40            Pro                                                             41            Glu                                                             42            Asp                                                             43            Ala/His                                                         44            Thr                                                             45            Gly                                                             46            Ala                                                             47            Asp                                                             48            Tyr                                                             49            Tyr                                                             50            Ser                                                             51            Ser                                                             52            --                                                              53            Arg                                                             ______________________________________                                    

Approximate yields of phenylthiohydantoin derivatives were calculatedfrom peak areas on the HPLC.

EXAMPLE 2

Following a procedure similar to the procedure of Example 1, and usingOrnithodoros moubata ticks obtained from a warthog burrow near LakeVictoria, Zimbabwe, (subspecies porcinus Walton) another polypeptidesequence having Factor Xa inhibitor activity such as that of TAP-1 wasdetermined. The other polypeptide was identified as TAP-2.

EXAMPLE 3

Starting with Boc-Ile-O-Pam resin, the alpha-amino Boc protecting group(tert-butylcarbonyl) is removed using trifluoracetic acid and methylenechloride, and the deprotected isoleucine neutralized withdiisopropylethyl amine. Boc-protected Cys (PMB) (where cysteine isprotected by p-methoxybenzyl) is then coupled to isoleucine mediated bydicyclohexylcarbodiimide, and deprotected with trifluoroacetic acid andmethylene chloride (protocol for Applied Biosystems Inc. peptidesynthesizer). Cys is then neutralized with diisopropylethylamine.Following this stepwise procedure of coupling withdicyclohexylcarbodiimide, deprotection with trifluoroacetic acid andmethylene chloride, and neutralization with diisopropylethylamine,Boc-protected Ala, Asn and the remaining 56 amino acids of the 60 aminoacid polypeptide are coupled in succession. The various amino acids maybe suitably protected in accordance with the usual peptide synthesispractice which is well known to those skilled in the art.

Cleavage of the peptide from the resin is achieved using HF/anisole(9:1(v/v)). To avoid alkylation of cysteine and tyrosine residues, athio-cresol and cresol scavenger mixture was used. The resin is washedwith ether, and 250 μM polypeptide concentration is immediately dilutedin about 2 liters of 0.1M acetic acid solution. The solution is thenstirred and its pH adjusted to about 8.0 using ammonium hydroxide.Purification is conducted using preparative HPLC in 0.1% TFA H₂ O-CH₃ CNgradient.

The finished amino acid sequence of the inhibitor is: ##STR2##

Inhibitory Activities of TAP-1 on Various Proteases

FIG. 3 shows the effect of increasing amounts of inhibitor on the ratioof the initial velocities in the presence (Vi) and absence (Vo) ofTAP-1. Because the TAP-1 was pre-incubated with the human Factor Xa toestablish equilibrium in the absence of substrate, a value for theintrinsic K_(i) irrespective of the mechanism was calculated (solidline) using the equation for tight binding inhibition described in thearticle by Morrison, J. F. in Biochem. Biophys. Acta 185,269 (1969). Theeffect on TAP-1 on various proteases also was tested. At a 300-foldmolar excess of TAP-1 over each protease, no inhibition of Factor VIIathrombin, chymotrypsin, elastase, trypsin, kallikrein, urokinase,plasmin, or S. aureus V8 protease was detected. The K_(i) was 0.58 nM.The inclusion of 1-5 mM Ca⁺⁺ had no effect on the ability of TAP-1 toinhibit Factor Xa. The effect of TAP-1 on clotting time in the presenceof prothrombin, activated partial thromboplastin and modified stypven isshown in Table II.

                  TABLE II                                                        ______________________________________                                        Effect of TAP-1 on Various                                                    Plasma Based Clotting Assays                                                                Clotting Time (Sec.)                                            ______________________________________                                        Prothrombin Time (Normal Human Plasma)                                        (control)     14.1                                                            (control      15.1                                                            1.15 pMol     15.1                                                            28.9 pMol     28.9                                                            57.8 pMol     31.2                                                            115 pMol      48.7                                                            Activated Partial Thromboplastin Time (Normal                                 Human Plasma)                                                                 (control)     32.6                                                            (control)     29.6                                                            (control)     31.6                                                            28.9 pMol     45.2                                                            57.8 pMol     57.7                                                            115 pMol      92.2                                                            Modified Stypven Time (Normal Human Plasma)                                   (control)     10.1                                                            (control)     10.6                                                            28.9 pMol     17.7                                                            57.8 pMol     31.2                                                            115 pMol      65.1                                                            ______________________________________                                    

The potential exists, in the use of recombinant DNA technology, for thepreparation of various derivatives of proteins of the present invention,variously modified by resultant single or multiple amino acidsubstitutions, deletions, additions or replacements, for example, bymeans of site directed mutagenesis of the underlying DNA. All suchallelic variations and modifications resulting in these derivatives areincluded within the scope of this invention so long as the essential,characteristic Factor Xa inhibitory activity of these proteins remainsunaffected in kind. The proteins are prepared (1) having methionine asits first amino acid (present by virtue of the ATG start signal codoninsertion in front of the structural gene) or (2) where the methionineis intra-or extracellularly cleaved, having its normally first aminoacid, or (3) together with either its signal polypeptide or a conjugatedprotein other than the conventional signal polypeptide, the signalpolypeptide or conjugate being specifically cleavable in an intra- orextracellular environment (see British Patent Application PublicationNo. 2,007,676A), or (4) by direct expression in mature form without thenecessity of cleaving away any extraneous, superfluous polypeptide. Thelatter is particularly important where a given host may not, or notefficiently, remove a signal peptide where the expression vehicle isdesigned to express the protein together with its signal peptide. In anyevent, the thus produced protein, in its various forms, is recovered andpurified to a level fitting it for use in inhibiting Factor Xa.

Exemplified below is a recombinant technique for producing the inhibitorin naturally folded and biologically active form.

EXAMPLE 4 Expression of the Inhibitor in Yeast

A recombinant gene encoding the inhibitor was synthesized andconstructed based on the primary amino acid sequence of TAP-1. Theproperly modified synthetic gene was inserted into a yeast expressionvector that allows for secretory expression. Yeast cells weretransformed with the vector containing the synthetic gene.

Because the amino acid sequence of TAP-1 was identified, appropriatelychosen synthetic oligonucleotides were used to construct the geneencoding the inhibitor. Eight oligonucleotides were synthesized, and thesynthetic gene constructed by annealing and ligation. ##STR3##

Each oligonucleotide was purified by electrophoresis on a 15%polyacrylamide gel, isolation and electroelution. Oligonucleotides IIthrough VII were treated with polynucleotide kinase and annealed incomplementary pairs (III and IV) and (V and VI). Oligonucleotides I andVIII were annealed directly with kinased II and VII respectively. Theoligonucleotides were annealed in kinase reaction buffer by heating to80° C. for two minutes and slow cooling over the course of an hour. Thefour annealed oligonucleotide pairs were pooled and treated with T4ligase. The resulting product was digested with EcoRI. The product,representing the synthetic gene, was isolated as a 200 bp fragment afterelectrophoresis of the mixture on a 2% agarose gel, the identifiedfragment excised and electroeluted.

The DNA fragment representing the synthetic gene was ligated to pJC264(Gan, Z.-R. et al. (1989) Gene 79:159-166) which had been previouslydigested with EcoRI and treated with alkaline phosphatase to yieldplasmid 276-2E. The ligation mixture was used to transform competent E.coli (JM109 available from Stratagene, Calif., U.S.A.) Ampicillinresistant cells were obtained and selected for on ampicillin plates. Thecorrect insert sequence in resulting plasmid clones was confirmed by DNAsequence analysis.

The strategy used to assemble this synthetic gene is given below. Inaddition, the resulting open reading frame and its translation ispresented. ##STR4##

The synthetic gene was inserted into the yeast expression vector in thefollowing manner. One plasmid, 276-2E, was selected, and a polymerasechain reaction product was obtained in a reaction using theoligonucleotide primers: ##STR5## The inhibitor DNA was subjected topolymerase chain reaction (PCR) -effected amplification (see U.S. Pat.No. 4,800,159, column 2, lines 36-68, column 3, column 4, and column 5,lines 1-20, hereby incorporated by reference). The DNA strands were heatdenatured in the presence of primers that bind to each strand. Theprimers instructed DNA polymerase, which performs its replicationfunction, to copy a particular portion of the strand. The process wascontinued with a series of heating and cooling cycles, heating toseparate strands, and cooling to allow annealing and primer extensionforming copies of the desired sequences. The cycles were repeated togenerate more and more copies of the specific sequences. Throughamplification, the coding domain to which terminal restriction sites areappended was obtained.

The PCR product was used to generate pKH4•TAP.

Construction of pKH4α2

Construction of pKH4 is described in Schultz, et al., Gene 61 (1987)123-133, which is incorporated by reference. The plasmid pJC197 (Schultzet al. Gene 54 (1987) 113-123) is an E. coli-S. cerevisiae shuttlevector which contains a unique BamHI cloning site between the yeast MFα1pre-pro leader and transcriptional terminator, originally derived inKurjan and Herskowitz (1982) ibid. pJC197 was digested with EcoRI+PstI,and the 0.7-kb PstI-EcoRI fragment containing a portion of the MFα1pre-pro-leader, a three-frame translational terminator, and MFα1transcriptional terminator, was gel-purified. GAL10p was isolated fromYEP51 by digestion with Sau3A, flush-ending with Po1Ik, ligating withoctameric BamHI linkers, and digestion with Sa1I.

The resulting 0.5-kb BamHI-Sa1I fragment bearing GAL10p was gel purifiedand ligated to a 35-bp Sa1I-PstI synthetic oligodeoxynucleotide adapterencoding the first 11 bp of the MFα1 nontranslated leader plus the ATGand first 8 aa of the MFα1 pre-pro-leader. The resulting 0.5-kb fragmentwas digested with BamHI, gel-purified, and ligated together with theaforementioned 0.7-kb PstI-EcoRI fragment plus the 4.0 kb EcoRI-BamHIvector fragment derived from pBR322. The resulting plasmid, pKH207-1,contains GAL10p fused to the MFα1 pre-pro-leader plus BamHI cloningsite, translational termination codons, and MFα1 transcriptionalterminator. Upon digestion with EcoRI and partial digestion with BamHI,an expression cassette of GAL10p fused to the yeast MFα1 pre-pro-leader,a unique BamHI cloning site, translational termination codons in allthree reading frames, and MFα1 transcriptional terminator sequence wasinserted into the yeast shuttle vector pCl/1 (Rosenberg et al. Nature312 (1984) 77-80) which contains the yeast 2μ DNA sequence for stablepropagation of the plasmid in yeast at high copy number, to form pKH4.

A 213-bp BamHI-PstI fragment encoding aa 9-79 of the ppL was preparedfrom the plasmid pα2 (Bayne et al., Gene 66 (1988) 235-244). The plasmidpα2 contains a portion of the yeast MFα1 pre-pro sequence (79aa)modified at codons 80 and 81 to create a BamHI site 6 aa upstream fromthe KEX2 processing site. The region corresponding to codon 9 (PstI) ofthe ppL to the BamHI site of pKH4 was removed from pKH4 after digestionwith BamHI followed by partial digestion with PstI. The modified vector,pKH4α2 was prepared by replacement of this excised sequence with theBamHI-PstI fragment from pα2. Plasmid pKH4α2 contains the yeast GAL10promoter, a portion of the MFα1 pre-pro leader (79 aa), a three-frametranslational terminator and MFα1 transcriptional terminator, the yeastLEU2 gene, yeast 2μ sequences, pBR322 derived sequences, including theAp^(R) gene and the origin of DNA replication (ori).

Construction of pKH4•TAP

Polymerase chain reaction resulted in a blunt end fragment which wasregenerated in the usual fashion by digestion with BamHI. The correctfragment was obtained after electrophoresis on a 2% agarose gel,excision of the band and electroelution. The purified fragment wasligated with the yeast expression vector pKH4α2 (Jacobson, M. A. et al.(1989) Gene 85: 513-518) that had been previously digested with BamHIand treated with calf alkaline phosphatase (FIG. 4). The correctsequence of plasmid clones in the correct orientation was confirmed byDNA sequence analysis. Fusion products produced from pKH4•TAP areproteolytically processed by the Lys-Arg-cleaving endopeptidase (KEX2)encoded by the KEX2 gene and products are secreted into culture medium.KEX2 cleaves on the C-terminal side of Lys-Arg residues.

Transformation of DMY6

Diploid yeast strain DMY6 (Schultz, L. D. (1987) Gene 61: 123-133) wastransformed with pKH4•TAP using standard protocols (Hinnen et al. (1978)Proc. Natl. Acad. Sci. USA 75: 1929-1933). One isolate was chosen anddesignated S. cerevisiae MY 2030 9718P281-3. The isolate was depositedwith the American Type Culture Collection and identified ATCC No. 20984.

From plate containing yeast transformants, single colony isolates wereobtained. These isolates were grown in 5×CM leu medium (0.85% yeastnitrogen base without amino acids and ammonium sulfate, 1% succinicacid, 0.6% NaOH, 0.5% ammonium sulfate, 0.06% isoleucine, 0.06%phenylalanine, 0.04% lysine, 0.04% tryptophan, 0.025% tyrosine, 0.02%adenine, 0.02% uracil, 0.01% arginine, 0.01% histidine, 0.01%methionine, 0.04% FeCl₃.6H₂ O, 0.03% ZnSO₄ -7H₂ O and 4% glucose) at 30°C. Cells were grown by inoculating a frozen stock culture of 281-3 onleucine-minus agar media for 3 days at 28° C. prior to the inoculationof 500 ml of selective medium (5×CM Leu⁻). The 500 ml seed culture wasgrown in a 2-1 Ehrlenmeyer flask for 16-18 hr at 28° C. using a rotaryshaker at 300 rpm prior to its inoculation into 2.5 l of 5×CM Leu⁻ brothcontaining 4% (w/v) galactose in a 1.5 l Bio-Flo III fermentor (NewBrunswick Scientific). The fermentor was operated at 28° C., 900 rpm,2.5 l/min air for 110-120 hr.

All work was done at 2°-8° C. Yeast cells were separated from broth (8liters containing 1.69 gm. recombinant TAP-1) of yeast culture (strain281-3) secreting recombinant TAP-1 by diafiltering the liquid through a500,000 MWCO hollow fiber cartridge (Romicon, PM500, 5 ft) using anAmicon DC10 unit. The clarified broth was then diafiltered through a100,000 MWCO hollow fiber cartridge (Amicon, H5P100-43) untilapproximately 500 mL of liquid remained in the reservoir. The retainedliquid was diafiltered with 8 liters of 20 mM BIS-TRIS, pH 6.0. (BufferA). The effluent of the 100,000 MWCO fibers was adjusted to pH 6.0 anddiluted with 63 liters of cold buffer A. The diluted broth contained1.67 gm of recombinant TAP-1. The diluted fermentation broth was dividedinto two 40 liter portions and wach was pumped at 2 liters/hr ontoseparate columns of Q-Sepharose Fast Flow™ (Pharmacia: 14 cm×10 cm I.D.)that had been equilibrated with buffer A. After all the diluted brothhad been pumped onto the columns they were each washed with 8 liters ofBuffer A to finish eluting unbound materials. The bound proteins wereeluted with 16 liters linear gradient of 0-500 mM NaCl in Buffer A.Fractions of 400 mL were collected and monitored for A₂₈₀ and inhibitionof human Factor Xa. Recombinant TAP-1 eluted at 175 mM NaCl.

The fractions that contained recombinant TAP-1 were combined and dilutedwith four volumes of 50 mM sodium acetate, pH 4.0 (Buffer B) beforebeing pumped at 2 liters/hr onto a column of S-Sepharose Fast Flow™(Pharmacia; 24.5 cm×7.5 cm I.D.) that had been equilibrated with BufferB. When all the sample had been pumped onto the column it was washedwith 8 liters of Buffer B and then the bound proteins were eluted with a16 liters linear gradient of 0-400 mM NaCl in Buffer B. The fractionswere monitored for A₂₈₀ and human Factor Xa inhibitory activity andrecombinant TAP-1 eluted at 190 mM NaCl. The amount of recombinant TAP-1eluted from the column was 1.58 gm. The pooled fractions containinginhibitory activity were loaded directly onto a 4.5×30 cm prep-pak C₁₈HPLC column (Waters Associates) equilibrated with aqueous 0.1% (v/v) CF₃COOH at 50 ml/min using a Delta-Prep preparative HPLC system (WatersAssoc.). Following application of the sample at the same flow rate thecolumn was washed with aqueous 0.1% (v/v) CF₃ COOH for 5 min at 100ml/min prior to the development of a CH₃ CN gradient in aqueous 0.1%(v/v) CF₃ COOH from 0-40% (v/v) at 1%/min. The peak of absorbance at 280nm which eluted at a CH₃ CN concentration of approximately 31% (v/v) wascollected by hand and dried by lyophilization. Gel filtrationchromatography can be used as an alternative to reserve phase HPLC toprepare desalted recombinant peptide. For gel filtration, Sephadex™ G-10(Pharmacia) was swollen in Milli-Q™ H₂ O and packed into a 2.5 cm(I.D.)×93 cm glass column and 90 mL of a solution of recombinant TAP-1purified by S-Sepharose chromatography was chromatographed on it.Fractions of 12.5 mL were collected and monitored for A₂₈₀ andconductivity. Protein eluted in the V_(o) and was well separated fromthe salt. The recovery of recombinant TAP-1 was 100%. The material wasdried by lyophilization. The purified recombinant polypeptide preparedby either method was found to be greater than 99% pure by analyticalreverse phase C₁₈ HPLC, quantitative amino acid analysis and automatedamino-terminal sequence analysis. A typical specific content of therecombinant inhibitor was between 115 and 120 nmole/mg with ahomogeneous amino-terminus beginning with tyrosine. The electrophoreticmobility of recombinant TAP-1 was identical to the native inhibitor asassessed sodium-dodecyl sulfate polyacrylamide gel electrophoresis underreducing conditions. The Factor Xa inhibitory activity of therecombinant TAP-1 was similar to that shown in FIG. 3 with a K_(i)determined to be 0.2 nM.

Effectiveness of Recombinant TAP-1

An in vivo model of thromboplastin-induced clot formation similar to VanRyn-McKenna, et al., (1989) Thrombosis and Haemostasis 61:7-9 was usedto determine the efficacy of the Factor Xa inhibitor recombinant TAP-1.Rabbits were pretreated with an infusion of recombinant TAP-1 for 60minutes, at which time a segment of the jugular vein was isolated andoccluded. Thromboplastin and whole blood withdrawn from the carotidartery were injected into the segment in order to induce clot formation.The jugular vein remained occluded for 30 minutes before the clot wasremoved and quantitated.

Clot formation in rabbits receiving no anticoagulant pretreatment hadnormalized clot values of 29.2±3.4%. Clot formation with i.v. infusionsof recombinant TAP-1 of 7 μg/kg/min, 37 μg/kg/min and 64 μg/kg/min were27.0±7.5% and 7.9±2.0%, and 1.9±1.0% respectively. These doses did notsignificantly increase the activated partial thromboplastin time orprothrombin time.

TAP-1 MUTAGENESIS

Desired mutations of the TAP-1 open reading frame were prepared by oneof two basic approaches: polymerase chain reaction mutagenesis, andKunkel mutagenesis.

PCR Mutagenesis

The use of polymerase chain reaction catalyzed by Taq DNA polymerase forsite-specific mutagenesis is described in Kadowaki et al., Gene 76(1989) 161-166. Kadowaka et al. describe introduction of mismatches intothe oligos used to prime polymerase chain reactions (Saiki, et al.,Science 230 (1985) 1350-1354 and Saiki et al., Science 239 (1988)487-491) in order to adopt the method to site-directed mutagenesis.

PCR Mutagenesis-Near-terminal location mutations

Mutations occurring near the ends of the TAP-1 coding sequence weremodified through polymerase chain reaction (PCR) amplification of theTAP-1 template. Near-terminal location mutations include the TAP-1mutation at residue position 53 which results in replacement of argininewith asparagine. By adding oligomer primers (complementary to the endsof the DNA to be amplified) to the TAP-encoding DNA and heating thereaction mixture, the DNA strands separate and permit annealling of theprimers to the template. The primers bind opposite strands and, uponreaction with Taq polymerase (Cetus Perkin-Elmer) in a number of cyclesof extension, strand separation and reannealling, the region between theprimers is replicated. If one or more of the primers includes amismatched region, that mutation is incorporated into the reactionproduct. TAP mutants were created through this amplification using theappropriate codon changes within the mutagenic primer. The PCR productwas cleaved with BamH1, gel purified and then inserted into the pKH4α2at the BamH1 site.

PCR Mutagenesis-Non-terminal location mutations

A modification of this approach was used for mutations desired atnon-terminal location, occurring near unique Taq1 or Xhol restrictionsites: Non-terminal location mutations include the TAP-1 mutations atresidue positions 9 and 23 which result in replacement of arginine withasparagine, and the TAP-1 mutations at residue position 16 which resultsin replacement of aspartic acid with arginine. Primers were prepared toamplify the TAP coding sequences and incorporate the mutations asbefore. The primers extended far enough to include restriction sitesequences present in the TAP gene. Another set of primers were used toamplify remaining sequences of gene and the same restriction site. Theresulting fragments were digested with that enzyme and ligated,reforming the TAP gene complete with the mutation. This material waspurified and BamH1 digested for insertion in pKH4α2 as described above.

Kunkel Mutagenesis

Kunkel Proc. Natl. Acad. Sci. U.S.A. Vol. 82 (1985) 488-492, describessite-specific mutagenesis which takes advantage of a strong biologicalselection against the original, unaltered genotype. By using arelatively normal uracil-containing DNA template, prepared by standardprocedures (Sagher and Strauss Biochemistry 22 (1983) 4518-4526) aftergrowth on an Escherichia coli dut⁻ ung⁻ strain (Tye et al. Proc. Natl.Acad. Sci. U.S.A. 75 (1978) 233-237), site-specific mutagenesisprocedures are used to produce mutations at high efficiencies withoutselection and in a few hours.

Mutants were prepared using a modification of the Kunkel method in whichsingle strand p276-2E plasmid DNA was annealed with a complementaryprimer containing the desired mutagenic sequences. The Kunkel method wasspecifically employed to obtain a TAP-1 mutation at residue position 27which results in replacement of arginine by asparagine. This complex wasextended by DNA polymerase, covalently linked with DNA ligase and theproduct used to transform E. coli DH5 cells (Bethesda ResearchLaboratories). Mutation recovery was improved by selection against theparental strand using E. coli host CAG629 (dut-ung-BioRad) to preparethe singlestrand template. In presence of M13K07 helper phage(Pharmacia), one strand of the plasmid DNA is copied, packaged intophage capsids and secreted into the supernatant, permitting isolation ofthe single strand DNA. Because this strand contains uracil, it isdegraded when the mutagenesis reaction mix is used to transform E. coliDH5(Dut⁺ Ung⁺), leaving the mutagenized strand as template forreplication. The modified TAP sequences were transferred as a BamH1cassette to the pKH4α2 vector.

This method was also employed using the expression vector as thetemplate for the mutagenesis reaction.

Therapy

The protein inhibits coagulation by inhibiting the Factor Xa pathwayrather than by inhibition of thrombin. Factor Xa pathway inhibition isachieved by administering the protein, either by continuous intravenousadministration or bolus administration, in combination with a suitablepharmaceutical composition carrier e.g. saline, at a suitable pH, e.g.7.4, such that the composition achieves the desired effect of preventingFactor Xa from inducing formation of thrombin from prothrombin.

The proteinaceous substance of this invention having Factor X_(a)inhibition activity can, like many proteins/peptides, formpharmaceutically acceptable salts with any non-toxic, organic orinorganic acid. Illustrative inorganic acids which form suitable saltsinclude hydrochloric, hydrobromic, sulphuric and phosphoric acid andacid metal salts such as sodium monohydrogen orthophosphate andpotassium hydrogen sulfate. Illustrative organic acids which formsuitable salts include the mono, di and tricarboxylic acids.Illustrative of such acids are, for example, acetic, glycolic, lactic,pyruvic, malonic, succinic, trifluroacetic, glutaric, fumaric, malic,tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic,hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic andsulfonic acids such as methane sulfonic acid and 2-hydroxyethanesulfonic acid. Salts of the carboxy terminal amino acid moiety includethe non-toxic carboxylic acid salts formed with any suitable inorganicor organic bases. Illustratively, these salts include those of alkalimetals, as for example, sodium and potassium; alkaline earth metals,such as calcium and magnesium; light metals of Group IIIA includingaluminium; and organic primary, secondary and tertiary amines, as forexample, trialkylamines, including triethylamine, procaine,dibenzylamine, 1-ethenamine; N,N'-dibenzylethylenediamine,dihydroabietylamine, N-(lower)alkylpiperidine, and any other suitableamine.

The anticoagulant dose of the proteinaceous substance of this inventionhaving Factor X_(a) inhibition activity is from 0.2 mg/kg to 250 mg/kgof patient body weight per day depending on, for example, the patient,and the severity of the thrombotic condition to be treated. The suitabledose for a particular patient can be readily determined. Preferably from1 to 4 daily doses would be administered typically with from 5 mg to 100mg of active compound per dose. The concentration of the proteinaceoussubstance of this invention having Factor X_(a) inhibition activityrequired to inhibit Factor X_(a) when used to inhibit blood coagulationor Factor X_(a) in a medium such as stored blood can be readilydetermined by those skilled in the art.

Anticoagulant therapy is indicated for the treatment and prevention of avariety of thrombotic conditions, particularly coronary artery andcerebrovascular disease. Those experienced in this field are readilyaware of the circumstances requiring anticoagulant therapy. The term"patient" used herein is taken to mean mammals such as primates,including humans, sheep, horses, cattle, pigs, dogs, cats, rats, andmice. Inhibition of Factor X_(a) is useful not only in the anticoagulanttherapy of individuals having thrombotic conditions, but is usefulwhenever inhibition of blood coagulation is required such as to preventcoagulation of stored whole blood and to prevent coagulation in otherbiological samples for testing or storage. Thus, the proteinaceoussubstance of this invention having Factor X_(a) inhibition activity canbe added to or contacted with any medium containing or suspected ofcontaining Factor X_(a) and in which it is desired that bloodcoagulation be inhibited.

Although the proteinaceous substance of this invention having FactorX_(a) inhibition activity may survive passage through the gut followingoral administration, applicants prefer non-oral administration, forexample, subcutaneous, intravenous, intramuscular or intraperitoneal;administration by depot injection; or by implant preparation.

For parenteral administration the proteinaceous substance of thisinvention having Factor X_(a) inhibition activity may be administered asinjectable dosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier whichcan be a sterile liquid such as water and oils with or without theaddition of a surfactant and other pharmaceutically acceptableadjuvants. Illustrative of oils which can be employed in thesepreparations are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, and mineral oil. Ingeneral, water, saline, aqueous dextrose and related sugar solutions,ethanol and glycols such as propylene glycol or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.

The proteinaceous substance of this invention having Factor X_(a)inhibition activity can be administered in the form of a depot injectionor implant preparation which may be formulated in such a manner as topermit a sustained release of the active ingredient. The activeingredient can be compressed into pellets or small cylinders andimplanted subcutaneously or intramuscularly as depot injections orimplants. Implants may employ inert materials such as biodegradablepolymers or synthetic silicones, for example, Silastic, silicone rubberor other polymers manufactured by the Dow-Corning Corporation.

The protein may be used alone or in combination with other proteins. Forexample, TAP enhances the efficiency of tissue plasminogenactivator-mediated thrombolytic reperfusion. TAP may be administeredfirst following thrombus formation, and tissue plasminogen activator orother plasminogen activator is administered thereafter.

Deposit

S. cerevisiae MY 2030 9718P281-3 deposited with the American TypeCulture Collection, 12301 Parklawn Drive Rockville, Md. 20852, USA, isdesignated ATCC 20984. The deposit was made Feb. 21, 1990 under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure andthe Regulations thereunder (Budapest Treaty). Maintenance of a viableculture is assured for 30 years from date of deposit. The organisms willbe made available by ATCC under the terms of the Budapest Treaty, andsubject to an agreement between Applicants and ATCC which assuresunrestricted availability upon issuance of the pertinent U.S. patent.Availability of tye deposited strains is not to be construed as alicense to practice the invention in contravention rights granted underthe authority of any government in accordance with its patent laws.

What is claimed is:
 1. A purified and isolated protein having thesequence: ##STR6## wherein AA⁹ is Arg or Asn,AA²³ is Arg or Asn, AA²⁷ isArg or Asn, and AA⁵³ is Arg or Asn.
 2. A protein of claim 1, wherein AA⁹is Arg, AA²³ is Arg, AA²⁷ is Arg, and AA⁵³ is Arg.
 3. A protein of claim1, wherein AA⁹ is Asn, AA²³ is Arg, AA²⁷ is Arg, and AA⁵³ is Arg.
 4. Aprotein of claim 1, wherein AA⁹ is Arg, AA²³ is Arg, AA²⁷ is Arg, andAA⁵³ is Arg.
 5. A protein of claim 1, wherein AA⁹ is Arg, AA²³ is Arg,AA²⁷ is Asn, and AA⁵³ is Arg.
 6. A protein of claim 1, wherein AA⁹ isArg, AA²³ is Arg, AA²⁷ is Arg, and AA⁵³ is Asn.
 7. A therapeuticcomposition for inhibiting blood coagulation Factor Xa comprising aneffective amount of the protein of claim 1 and apharmaceutically-acceptable carrier.
 8. A method for inhibiting bloodcoagulation Factor Xa in a mammal comprising administering to the mammalan effective dose of a composition of claim
 7. 9. A method for achievingthrombolytic reperfusion following thrombus formation in a patientcomprising administering a protein of claim 1 to inhibit Factor Xa andthereafter administering tissue plasminogen activator or otherplasminogen activator.